Genome instability is a hallmark of cancer. Replication stress (RS) results in genome instability by interfering with fork progression during DNA replication and thus is a potent inducer of a variety of hereditary and non- hereditary diseases including cancer. Therefore, the knowledge of the cellular responses to RS and the accurate elucidation of the mechanisms that stabilize the genome under RS are crucial for understanding cancer development and choosing the most efficient cancer therapy. While many proteins have been identified as important players in protecting genome stability under RS, how they interact with each other to preserve fork stability and promote fork restart is unclear. In the last funding cycle, we have established that the human CTC1/STN1/TEN1 complex (the CST complex) is a new player in countering RS. We have published findings demonstrating that in response to RS, CST proteins form distinct foci that colocalize with RAD51. We have also determined that RS induces physical interaction between CST and RAD51, and that this interaction is regulated by ATR in a DNA-independent manner. In addition, CST assists in RAD51 recruitment to stalled forks, particularly those stalled at G-rich repetitive sequences. The Cancer Genomics Atlas database shows that CST genes are frequently altered in multiple types of cancers including melanoma, prostate, breast and uterine cancers, indicating a previously-unrecognized role of CST in cancer development. These significant findings, together with additional unpublished preliminary studies, support the central hypothesis proposed in this competitive renewal ? that CST antagonizes fork degradation and that its deficiency induces genome instabilities that promote tumorigenesis. We will test this hypothesis by characterizing the functional significance of the CST/RAD51 interaction in antagonizing nascent strand degradation (Aim 1), by determining how CST coordinates with BRCA2 to inhibit excessive nascent strand degradation at stalled forks (Aim 2), and by determining whether Stn1 deletion promotes RS-induced tumor formation (Aim 3). We will integrate contemporary technologies, including CRISPR/Cas9, the DNA fiber and iPOND assays, as well as ChIP-seq, to accomplish the goals of the proposed research. In addition, a new transgenic mouse model will be developed that will serve as a valuable tool for the scientific community to study the role of genome maintenance in disease development and treatment. It is expected that findings from the proposed research will reveal novel information regarding the rescue of stalled replication and the preservation of genome stability.